Nitrogen and Oxygen Isotope Signatures of Nitrogen Compounds during Anammox in the Laboratory and a Wastewater Treatment Plant

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Isotopic fractionation factors against ¹⁵N and ¹⁸O during anammox (anaerobic ammonia oxidization by nitrite) are critical for evaluating the importance of this process in natural environments. We performed batch incubation experiments with an anammox-dominated biomass to investigate nitrogen (N) and oxygen (O) isotopic fractionation factors during anammox and also examined apparent isotope fractionation factors during anammox in an actual wastewater treatment plant. We conducted one incubation experiment with high δ¹⁸O of water to investigate the effects of water δ¹⁸O. The N isotopic fractionation factors estimated from incubation experiments and the wastewater treatment plant were similar to previous values. We also found that the N isotopic effect (¹⁵ε_NXR of –77.8 to –65.9‰ and ¹⁵Δ_NXR of –31.3 to –30.4‰) and possibly O isotopic effect (¹⁸ε_NXR of –20.6‰) for anaerobic nitrite oxidation to nitrate were inverse. We applied the estimated isotopic fractionation factors to the ordinary differential equation model to clarify whether anammox induces deviations in the δ¹⁸O vs δ¹⁵N of nitrate from a linear trajectory of 1, similar to heterotrophic denitrification. Although this deviation has been attributed to nitrite oxidation, the O isotopic fractionation factor for anammox is crucial for obtaining a more detailed understanding of the mechanisms controlling this deviation. In our model, anammox induced the trajectory of the δ¹⁸O vs δ¹⁵N of nitrate during denitrification to less than one, which strongly indicates that this deviation is evidence of nitrite oxidation by anammox under denitrifying conditions.